Recent studies have begun to elucidate the role of the HIV-1 encoded viral infectivity factor (Vif)in neutralizing a potent antiretroviral system that occurs in lymphocytes and macrophages. This system principally involves the cytidine deaminases APOBEC3G (A3G) and/or ASF, which are incorporated intoHIV- 1 cores where they lethally hypermutate newly synthesized viral reverse transcripts. Vif binds to A3G and A3F and induces their polyubiquitination and degradation, thereby eliminating them from infected cells and precluding their incorporation into HIV-1 progeny. The additional APOBEC3 paralogs ASA, A3B, and A3C have weaker anti-HIV-1 activities in standard assays, and it is believed that their concentrations might also be relatively low in T cells and macrophages. However, new evidence suggests that APOBEC3 expression levels and activities can be altered dramatically by extracellular signals and by cell-specific factors. Therefore, these other APOBEC3 paralogs might potentially influence HIV-1 replication in specific cells or compartments or during inflammation in some patient tissues. Recently, we found that HIV-1 Vifs use nonidentical sites to promiscuously bind to all APOBEC3 paralogs and that natural Vif variants degrade diverse APOBECSs with highly distinctive specificities in a standard assay system. Several mutations in Vif that eliminate its binding to A3G do not eliminate its binding to ASF, suggesting that these key cytidine deaminases associate differently with Vif.These and additional recent insights raise important questions concerning the mechanisms for Vif binding to ASF and A3G, the roles of Vif diversity in patients, and the identities and functions of cellular factors that associate with A3G and ASF to control their anti-HIV-1 activities. In addition, we have recently succeeded in producing large amounts of Vif and A3G in Pichia pastoris yeast, which enables structural investigations. Based on these considerations, we propose three substantive and synergistic aims: (1) Optimize large-scale production and purification of soluble Vif, A3G, and ASF. Analyze Vif homooligomerization, determine whether Vif associates directly with A3G and/or ASF, and use these purified proteins in collaborative physical and structural investigations. Employ site-directed mutagenesis to further analyze these protein interactions. (2) Analyze the role of adaptive Vif variance in HIV-1 replication in patients and in cell cultures. (3) Use tandem affinity purification and electrospray mass spectrometry to identify cellular proteins and RNAs that associate with A3G, and analyze the effects of these proteins and RNAs on anti-HIV-1 activities of A3G and ASF. This program substantively addresses important issues concerning Vif and APOBEC3 diversities and provides unique approaches and resources for analyzing their roles in HIV-1 replication and pathogenesis.